This is an application for a K08 Clinical Scientist Mentored Career Development Award for Dr. Jeffrey Cheng, a Dermatologist at the University of California, San Francisco. The candidate's long-term goal is to become a leading independent investigator of epigenetic mechanisms in epidermal biology and disease. This grant will be invaluable in supporting Dr. Cheng's transition towards intellectual independence and direction of his own research program. Specifically, this K08 award will support an intensive 5-year training plan to develop a strong scientific knowledge base and new technical skills in computational biology, epithelial biology, and epigenetics/epigenomics and to improve grant and scientific writing skills. Dr. Joseph Costello will mentor Dr. Cheng's scientific training and carer development. Dr. Costello, a Professor of Neurosurgery at UCSF and Director of a NIH Roadmap Epigenome Mapping Center, is a leading epigenomics researcher who studies epigenetic mechanisms in brain cancer and development. Dr. Ting Wang, a computational biologist with expertise in analyzing, integrating, and displaying deep sequencing-based epigenomics data, and Dr. Theodora Mauro, who studies epidermal differentiation and barrier function, will co-mentor Dr. Cheng. The proposed research seeks to understand the role of DNA methylation and hydroxymethylation in epidermal homeostasis. While DNA methylation clearly affects epidermal homeostasis, its gene regulatory role outside of promoters remains unclear. The role of DNA hydroxymethylation in the epidermis has not been studied. Emerging evidence suggests a significant role for DNA methylation and hydroxymethylation at enhancers. Consistent with this, preliminary DNA methylation data reveals that the majority of hypomethylated regions in proliferating keratinocytes overlap with enhancers. To explore the epidermal role of DNA methylation at enhancers and DNA hydroxymethylation, Dr. Cheng will perform next generation sequencing-based epigenetic profiling assays to identify differentially methylated and hydroxymethylated regions between proliferating and differentiating keratinocytes. This will be followed by experiments to validate keratinocyte- relevant candidate enhancers identified by their DNA methylation or hydroxymethylation patterns and assess the functional consequences of epidermal DNA hydroxymethylating enzyme and 5-hyroxymethylation depletion. These data will serve as a foundation to help achieve Dr. Cheng's long-term goal of understanding how epigenetic mechanisms are dysregulated in epidermal disease.